Bisphosphonate derivatives, their preparations and uses

ABSTRACT

The present invention relates to derivatives of 1-hydroxymethylene-1,1-bisphosphonic acid, the pharmaceutical compositions comprising them, and their application in therapeutics, particularly for the treatment of cancerous tumors. It is also directed to a methods of preparation of such derivatives.

FIELD OF THE INVENTION

[0001] The present invention relates to new derivatives of1-hydroxymethylene-1,1-bisphosphonic acid or bisphosphonate, thepharmaceutical compositions comprising them, and their application intherapeutics, particularly for the treatment of cancerous tumors andviral or inflammatory hepatic diseases. It also discloses methods ofpreparation of such derivatives.

BACKGROUND OF THE INVENTION

[0002] Derivatives of 1-hydroxymethylene-1,1-bisphosphonic acid displayremarkable antitumoral properties and their medical applications havebeen therefore the subject of in-depth research. Such derivatives,characterized by a P—C—P bond, are stable analogs of pyrophosphate andare resistant to enzymatic hydrolysis. Regarding the therapeuticapplications of diphosphonic acid derivatives, it is well known thatvarious derivatives have properties useful in the treatment ofinflammation, osteoporosis or some bone metastasis. In particular, theyare used for their ability to inhibit bone resorption to treat numerousdiseases characterized by abnormal calcium metabolism. Bone resorptionis pathologically increased in metastasis of certain types of cancersuch as breast or prostate cancer, and is accelerated in different formsof osteoporosis including that related to age. Patients with this typeof metastasis can now benefit from treatment regimens includingbisphosphonates (Diel et al., 2000; Lipton, 2000; Mincey et al., 2000).It should be pointed out that bone is the third most common site ofmetastasis and that over 80% of patients who die from cancer have bonetumors at autopsy.

[0003] A number of diphosphonic acid derivatives and their propertiesuseful in various applications have been described in the literature.

[0004] For example, didronic acid has been known for years as active fordrugs in the treatment of bone diseases such as osteoporosis, and moreparticularly disodium didronate described in French patent 8,441 M. Aderivative is described for example in U.S. Pat. No. 4,705,561 relatingto alendronic acid which inhibits bone resorption and which can be usedin the treatment of osteoporosis. Another similar structure is describedin British patent 2,312,165 relating to ibandronic acid havinganti-inflammatory properties.

[0005] Alkane-1,1-diphosphonic acid with an amino-acid, and possessingantitumor and bone resorption activities have been described in PCTpatent application WO 97/49711. Other diphosphonic acid derivativescomprising a phenyl substituent at the 1 position are described in U.S.Pat. No. 4,473,560 which discloses their anti-inflammatory activity,more particularly a antiarthritic activity, or in PCT patent applicationWO 97/04785 relating to phenol substituted diphosphonates havingantineoplasic activity. Further, European patent EP 537,008 describesdiphosphonate derivatives having a lipophilic group, which are useful inthe preparation of a medicament inhibiting protein prenyl transferase,likely to block the neoplasic transformation resulting from rasoncogenes.

[0006] This anti-osteoclastic action of bisphosphonates is postulated tooccur by induction of apoptosis in osteoclasts (Luckman et al., 1998)via inhibition of the mevalonate pathway and of cholesterol synthesis.

[0007] Bisphosphonates inhibit in vitro the proliferation of breasttumor cells (Fromigue et al., 2000; Hiraga et al., 2001; Jagdev et al.,2001; Senaratne et al., 2000; Yoneda et al., 2000) and prostate tumorcells (Lee et al., 2001). This in vitro inhibition is due to apoptosisof tumor cells and is accompanied by expression of the bcl-2 gene(Senaratne et al., 2000) and activation of caspases (Fromigue et al.,2000).

[0008] Besides the hereinabove effects, bisphosphonates may have severaladditional actions, such as inhibition in vitro of adhesion of breasttumor cells to bone matrices (Boissier et al., 1997; Van der Pluijm etal., 1996), induction in vitro of myeloma cell apoptosis (Shipman etal., 1997, 1998, 2000a) or inhibition of the activity (but not theproduction) of matrix metalloproteinases in breast or prostate carcinomacells (Boissier et al., 2000; Ichinose et al., 2000; Teronen et al.,1997).

[0009] Bisphosphonates have also been utilized in the treatment oflymphoblastic leukemia (Ogihara et al., 1995; Takagi et al., 1998).Leukemic cells induce angiogenesis in bone marrow, this being necessaryfor their proliferation. Treatment of lymphoblastic leukemia withbisphosphonates is accompanied by a significant decrease in angiogenesis(Perez-Atayde et al., 1997).

[0010] Recent data point toward the very likely involvement ofangiogenic factors in the formation of bone metastases. For instance, ina study in an in vivo murine model of experimental breast tumor cellmetastasis (MDA MB 231), Van der Pluijm et al. (2001) hypothesized thatelevated expression of angiogenic (VEGF) and osteolytic (PTH) factors inthe tumor cells is involved in osteotropism and bone loss of bonemetastases.

[0011] Although the antiproliferative activity of bisphosphonates ontumor cells in vitro is now well established, the question of whetherbisphosphonates can exert antitumoral action in vivo is still open. Somedata appear to argue for an antiproliferative action of bisphosphonatesin vivo, at metastatic sites in bone. For instance, according to Hiragaet al. (2001), bisphosphonates would diminish the tumor burden ofmetastatic cells in bone. However, to our knowledge, no data have beenreported on an antitumoral action of bisphosphonates in vivo on primarytumors.

[0012] Bisphosphonates are metabolized by the body to a low extent andthe active fraction represents only 3 to 7% of the absorbed dose. Thislow bioavailability of bisphosphonates after oral administration resultsfrom their low lipophilicity (Lin, 1996) which is due to their highstate of ionization at physiologic pH. Their absorption is furtherreduced by the strong negative charge and fairly large size of thesemolecules (Ruifrok and Mol, 1983; Pade and Stavchanvsky, 1997).Moreover, the absorption of bisphosphonates is reduced further still bytheir high level of complexation with calcium and other divalent ions inthe intestine (Lin, 1996). Their administration often causesgastrointestinal symptoms and other side effects (Adami and Zamberlan.,1996; Mondelo et al., 1997).

[0013] Moreover, drug acquired resistance is now a major concern in thecancer therapy, and most of human cancers are resistant to the effectsof chemiotherapies.

[0014] To improve their therapeutic effects, various approaches havebeen proposed. The first consists in the use of a peptide vector graftedto the side chain of hydroxybisphosphonic acid (Ezra et al., 2000).Other studies suggest encapsulating the drug in microspheres (Patashniket al., 1997) or in liposomes (Ylitalo et al., 1998).

[0015] The present invention is therefore directed at providing newbisphosphonate derivatives with improved bioavailability andsatisfactory therapeutic efficacy.

[0016] Two methods of synthesis described in the literature yield1-hydroxymethylene-1,1-bisphosphonic acids.

[0017] In the first, the desired products are obtained in a single step.This method consists in heating a mixture of carboxylic acid in thepresence of phosphorous acid and phosphorus trichloride at 100° C. forseveral hours.

[0018] The conditions of this reaction have been studied in greatdetail. In fact, since 1970, more than fifty patents and articles havebeen published. The drawbacks of this method, however, are numerous.Indeed, the drastic operating conditions are not suited to labilesubstrates. Furthermore, extraction of the bisphosphonate from thereaction medium is often tricky.

[0019] The second procedure is an indirect method involving synthesis of1-hydroxymethylene-1,1-bisphosphonic esters followed by a dealkylationstep. The α-ketophosphonates are typically prepared via a MichaelisArbuzov reaction, starting from a phosphite with the structure P(OR)₃and an acid chloride. The bisphosphonic ester is then normally obtainedby reaction of the α-ketophosphonate with a dialkylphosphite HOP(OR)₂.

[0020] The dealkylation step is carried out either by hydrolysis inhydrochloric acid or by treatment with bromotrimethylsilane followed bymethanolysis.

[0021] Unfortunately, neither of these two synthetic methods yieldspartially esterified 1-hydroxymethylene-1,1-bisphosphonic acids.

[0022] The present invention therefore equally proposes methods ofpreparation of bisphosphonates allowing regioselective addition of oneor more ester functions on the phosphonic acid moieties.

SUMMARY OF THE INVENTION

[0023] Therefore, one object of the present invention comprises thebisphosphonate derivatives represented by the following formula (I) or(I′):

[0024] wherein R₁, R₂ and R₃, which are the same or different, eachrepresent a hydrogen atom, an alkyl, aryl, acyloxyalkyl or heterocyclegroup, and R′ and R″, which are the same or different, each represent ahydrogen atom, an alkali metal or alkaline earth atom,

[0025] A represents a group with the formula —CH₂)n-R₄,

[0026] R₄ represents a hydrogen atom, an alkyl group, an aryl group, aheterocycle or a group with the formula —NR₅R₆,

[0027] R₅ and R₆, which are the same or different, represent a hydrogenatom or an alkyl group,

[0028] n is a whole number from 0 to 24 inclusive, with the exception ofcompounds represented by formula (I) in which R₁, R₂ and R₃simultaneously represent a hydrogen atom, and compounds represented byformula (I′) in which R′ and R″ simultaneously represent a hydrogenatom, their optical and geometrical isomers, their racemates, theirsalts, their hydrates and their mixtures.

[0029] The present invention provides therefore compounds which presentadvantageously a good bioavailability, in particular when compared tocompounds already used or described as therapeutic agents, such as forinstance Etidronate (Procter & Gamble), Alendronate (Merck), Pamidronate(Novartis), Olpadronate (Gador), Ibandronate (Borhinger-Mannheim),compound EB1053 (Leo), compound YH 529 (Aventis) or Residronate (Procter& Gamble).

[0030] Another object of the present invention is a pharmaceuticalcomposition comprising at least one compound represented by formula (I)or (I′) such as described hereinabove, possibly in association withanother therapeutically active substance, in a pharmaceuticallyacceptable support.

[0031] It further relates to a method of treating a disordercharacterized by abnormal calcium metabolism, such as cancers orosteoporosis, comprising administering to a patient in need of suchtreatment a therapeutically effective amount of at least one compoundrepresented by formula (I) or (I′).

[0032] Another object of the present invention is a method for treatingviral or inflammatory hepatic disease, comprising administering to apatient in need of such treatment a therapeutically effective amount ofat least one compound represented by formula (I) or (I′).

[0033] A final object of the present invention is methods for producingcompounds represented by formula (I) or (I′).

LEGEND TO THE FIGURES

[0034]FIG. 1: Reaction diagrams of the method of preparation ofcompounds according to the invention. A is defined as hereinabove.

[0035]FIGS. 2 and 3: Inhibition in vitro of the proliferation of A 431tumor cells by BP1 (FIG. 2) and BP2 (FIG. 3) respectively, after 24, 48and 72 h of culture. Results are expressed as number of cells±SE (bars).*P<0.05; **P<0.01 compared to controls.

[0036]FIG. 4: Inhibition in vivo of the growth of A 431 tumors by BP1and BP2. Implantation of A431 cells in nude mice induces formation ofsubcutaneous tumors. One week after injection of the cells, the tumorswere treated with BP1 and BP2 at doses of 0.006, 0.06 and 0.6 mg/mouse,twice a week for 5 weeks. Results are expressed as tumor volume±SE(bars). *P<0.05 compared to controls.

[0037]FIG. 5 : Immunohistochemical analysis of angiogenesis of A 431tumors. Endothelial cells were detected with a specific marker (GSL 1).Results are expressed as area±SE (bars) of endothelial cells quantifiedby image analysis (NIH image). *P<0.01 compared to controls.

DETAILED DESCRIPTION OF THE INVENTION

[0038] Therefore, one object of the present invention comprises thebisphosphonate derivatives represented by the following formula (I) or(I′):

[0039] wherein R₁, R₂ and R₃, which are the same or different, eachrepresent a hydrogen atom, an alkyl, aryl, acyloxyalkyl or heterocyclegroup, and R′ and R″, which are the same or different, each represent ahydrogen atom, an alkali metal or alkaline earth atom,

[0040] A represents a group with the formula —CH₂)n-R₄,

[0041] R₄ represents a hydrogen atom, an alkyl group, an aryl group, aheterocycle or a group with the formula —NR₅R₆,

[0042] R₅ and R₆, which are the same or different, represent a hydrogenatom or an alkyl group,

[0043] n is a whole number from 0 to 24 inclusive, with the exception ofcompounds represented by formula (I) in which R₁, R₂ and R₃simultaneously represent a hydrogen atom, and compounds represented byformula (I′) in which R′ and R″ simultaneously represent a hydrogenatom, their optical and geometrical isomers, their racemates, theirsalts, their hydrates and their mixtures.

[0044] The alkyl, aryl, heterocycle or acyloxyalkyl groups describedhereinabove are possibly substituted by at least one group chosen fromamong an aryl group, a heterocycle group, a heterocycloalkyl group, analkyl group, an alkenyl group, an alkynyl group, an alkylthio group, ahalogen atom, preferably Cl, F, Br, a hydroxyl group, an NO₂ group, analkoxy group, an ester group (—COOR), an amino group —NRR′″, an acidmoiety, an amide group (—CONHR or —NHCOR), wherein R and R′″ represent,independently of each other, a hydrogen atom, an alkyl, aryl, heteroarylor acyloxyalkyl group.

[0045] In the context of the present invention, the term “alkyl” morespecifically means a linear, branched or cyclic hydrocarbon group having1 to 24, preferably 1 to 12, carbon atoms such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, n-hexyl.C₁-C₆ groups are especially preferred. Methyl and ethyl groups are moreespecially preferred. The alkyl group may possibly be interrupted by oneor more heteroatoms, preferably N, S, O or P.

[0046] The term <<alkenyl>> refers to a hydrocarbon group having atleast one unsaturated ethylene bond and the term <<alkynyl>> refers to ahydrocarbon group having at least one unsaturated acetylene bond.

[0047] The <<aryl>> groups are mono-, bi- or tri-cyclic aromatichydrocarbons having from 6 to 18 carbon atoms. Examples include aphenyl, α-naphthyl, β-naphthyl or anthracenyl group, in particular.

[0048] <<Alkoxy>> groups correspond to the alkyl groups definedhereinabove bonded to the rest of the compound by an —O— (ether) bond.

[0049] <<Alkylthio>> groups correspond to the alkyl groups definedhereinabove bonded to the rest of the compound by an —S— (thioether)bond.

[0050] <<Acyloxyalkyl>> groups are acyloxy groups bonded to the rest ofthe compound by an alkyl chain (C1-24), preferably C1-4. Acyloxymethyland pivaloyloxymethyl groups are specific examples.

[0051] <<Halogen>> is understood to mean a fluorine, chlorine, bromineor iodine atom.

[0052] <<Heteroatom>> is understood to mean an atom chosen from among O,N and S.

[0053] Arylalkyl (or aralkyl) groups are groups comprising an arylfunction as defined hereinabove bonded to the rest of the compound bymeans of an alkyl chain.

[0054] <<Heterocycle>> or <<heterocycloalkyl>> groups are groupscontaining 5 to 18 rings comprising one or more heteroatoms (generallyN, O, S or P), preferably 1 to 5 endocyclic heteroatoms. They may bemono-, bi- or tri-cyclic. They may be aromatic or not. Preferably, andmore specifically for R₅, they are aromatic heterocycles. Examples ofaromatic heterocycles include pyridine, pyridazine, pyrimidine,pyrazine, furan, thiophene, pyrrole, oxazole, thiazole, isothiazole,imidazole, pyrazole, oxadiazole, triazole, thiadiazole and triazinegroups. Examples of bicycles include in particular quinoline,isoquinoline and quinazoline groups (for two 6-membered rings) andindole, benzimidazole, benzoxazole, benzothiazole and indazole (for a6-membered ring and a 5-membered ring). Nonaromatic heterocyclescomprise in particular piperazine, piperidine, etc.

[0055] The alkaline metal atoms include sodium and potassium. Thealkaline earth atoms include calcium.

[0056] In formula (I′) as defined above, R′ and/or R″ representpreferably an hydrogen atom or sodium, calcium or potassium atom.

[0057] The preferred compounds are represented by formula (I) whereinR₁, R₂ and R₃ are a hydrogen atom or an alkyl group of C1-12, moreparticularly C1-6.

[0058] Advantageously, at least two of the substituents R₁, R₂ and R₃are different from a hydrogen atom.

[0059] In an especially preferred variant of the invention, thesubstituents R₁, R₂ and R₃, which are different from a hydrogen atom,are identical.

[0060] The preferred compounds are represented by formula (I) whereinR₁, R₂ and R₃ represent a methyl or ethyl group.

[0061] The preferred compounds are represented by formula (I) or (I′) inwhich A represents a group with formula —CH2)n-R₄, wherein n is a wholenumber from 1 to 12 inclusive, preferably from 1 to 6 inclusive,advantageously from 1 to 3 inclusive.

[0062] The preferred compounds are represented by formula (I) or (I′) inwhich R₄ is a C1-C6 alkyl group, a heterocycle, a phenyl group, or agroup with formula —NR₅R₆, in which R₅ and R₆, which are the same ordifferent, represent a hydrogen atom or an alkyl group of C1 to C6,wherein n is advantageously a whole number from 1 to 6 inclusive,preferably from 1 to 3 inclusive.

[0063] According to a particular embodiment of the invention, thepreferred compounds have the formula (I) or (I′) wherein A is a groupchosen from:

[0064] In a particular embodiment, compounds of formula (I′) arecompounds wherein A represents

[0065] The compounds according to the invention, and in particularcompounds of formula (I), have the advantage of improved bioavailabilityand therefore a highly satisfactory therapeutic effect. In fact, withoutascribing to any particular theory of the invention, the ester bonds ofthe compounds according to the invention increase the lipophilicity ofthe latter and in this manner in particular the compounds appear to moreefficiently cross the gastrointestinal barrier when administered orally,the ester bonds then being lysed to release the correspondingbisphosphonic acid.

[0066] The subject of the present invention relates also to methods ofpreparation of compounds represented by formula (I) and (I′).

[0067] These methods of preparation have many advantages. They aresimple to implement on an industrial scale and produce high yields ofbisphosphonates.

[0068] The method for preparing compounds of formula (I) disclosed inthe present invention comprises the following steps:

[0069] contacting (or reacting) at least one acid halide represented byformula (II): ACOX, or one α-ketophosphonate represented by formula(III):

[0070] with at least one silyl phosphite represented by formula (IV):

P[(OSialk₃)_(x)][OR₃]_(3-x)  (IV)

[0071] wherein R₁, R₂ and R₃, which are the same or different, representan alkyl, aryl, acyloxyalkyl or heterocycle group,

[0072] A being defined hereinabove,

[0073] X represents a halogen atom, preferably chlorine,

[0074] alk is a C1-6 alkyl group,

[0075] x is 2 or 3,

[0076] hydrolysis of the compounds obtained in the previous step.

[0077] The C1-6 alkyl group is such as defined hereinabove.

[0078] The halogen atom may be a chlorine, bromine, iodine or fluorineatom, preferably X is a chlorine atom.

[0079] When R₁ and R₂ are different from a hydrogen atom, the method ofpreparation of compounds represented by formula (I) advantageouslycomprises the following steps:

[0080] contacting at least one acid halide represented by formula (II):ACOX with at least one phosphite represented by formula (V):P(OR1)(OR2)(OR), wherein R₁, R₂ and R, which are the same or different,represent an alkyl, aryl, acyloxyalky, or heterocycle group, to form anα-ketophosphonate represented by formula (III),

[0081] contacting the α-ketophosphonate obtained in the previous stepwith at least one silyl phosphite represented by formula (IV) as definedhereinabove,

[0082] hydrolysis of the compounds obtained in the previous step.

[0083] More specifically, when R₁, R₂ and R₃ are different from ahydrogen atom, the silyl phosphite represented by formula (IV) which ispreferably used corresponds to a compound represented by formula (IV)wherein x is equal to 2.

[0084] More specifically, when R₁ and R₂ are different from a hydrogenatom and R₃ is a hydrogen atom, the silyl phosphite represented byformula (IV) which is preferably used corresponds to a compoundrepresented by formula (IV) wherein x is equal to 3.

[0085] When R₁ is different from a hydrogen atom and R₂ is a hydrogenatom, the method for producing compounds represented by formula (I)advantageously comprises the following steps:

[0086] contacting at least one acid halide represented by formula (II):.ACOX; with at least one silyl phosphite represented by formula (IV):

P[(OSialk₃)_(x)][OR₃]_(3-x)  (IV)

[0087] wherein R₃ is an alkyl, aryl, acyloxyalkyl, or heterocycle group,

[0088] A being defined hereinabove,

[0089] X represents a halogen atom, preferably chlorine,

[0090] alk is a C1-6 alkyl group,

[0091] x is a whole number from 0 to 3,

[0092] hydrolysis of the compounds obtained in the previous step.

[0093] Generally, the contacting step with the silyl compound isadvantageously carried out under stoichiometric conditions. However,when one wishes to obtain compounds represented by formula (I) in whichR₁ and R₃ are different from a hydrogen atom and R₂ is a hydrogen atom,the method of preparation is advantageously carried out by reacting atleast 1 molar equivalent of at least one acid halide represented byformula (II): ACOX, with at least 2 molar equivalents of at least onesilyl phosphite represented by formula (IV).

[0094] The method of preparation of compounds represented by formula (I)when R₁ is different from a hydrogen atom and R₂ and R₃ represent ahydrogen atom, is advantageously carried out by contacting at least oneacid halide represented by formula (II): ACOX, with at least one silylphosphite represented by formula (IV) wherein x is equal to 2 andcontacting the product so obtained with at least one silyl phosphiterepresented by formula (IV) wherein x is equal to 3.

[0095] The hydrolysis in the method of preparation of compoundsrepresented by formula (I) is generally carried out in a solvent capableof donating protons, such as notably methanol or ethanol. Preferably,methanol is the solvent utilized.

[0096] The yields of compounds of formula (I) are virtuallyquantitative. Furthermore, this method has the advantage of implementingthe abovementioned steps successively in situ, without the need oftransferring products.

[0097] Generally speaking, the steps of the method according to theinvention may be carried out at room temperature (18-30° C.) and atatmospheric pressure. Of course, those skilled in the art can vary theseconditions, where necessary.

[0098] More specifically, it should be pointed out that reactions withsilyl phosphite may be exothermic, although without occurrence of sidereactions. This exothermic nature may make it necessary to carry outthese reactions at temperatures below room temperature, moreparticularly at temperatures between −70° C. and 20° C., especially whenusing nitrobenzoyl chloride.

[0099] The steps preceding the hydrolysis step in the method accordingto the invention may be carried out in bulk or in a solvent such asCHCl₃, THF, CH₃CN, CH₂Cl₂, or ether. In an advantageous manner, they arecarried out in bulk.

[0100] The hydrolysis step is advantageously carried out for 1 to 4hours, more specifically for about 2 hours. It is preferably carried outat room temperature, between 18 and 30° C.

[0101] After the hydrolysis step, the method according to the inventionpreferably comprises purification of the resulting compound representedby formula (1). Such purification is performed by any known means.Preferably, a purification is performed by at least two successivewashes in a suitable solvent, particularly ether.

[0102] The starting products of the method according to the inventionare commercially available products which are therefore easy to obtain.With regard to the phosphites, tris trimethylsilyl phosphite iscommercially available. The other phosphites may be prepared as follows.The dialkylphosphite is treated with a concentrated ammonia solution at0° C. After evaporation of the water, the resulting salt is treated withhexamethyldisilazane for 4 hours under reflux. The reaction is outlinedbelow.

[0103] Reaction diagrams of the method according to the invention aredepicted in FIG. 1.

[0104] According to the present invention, phenylacetic bisphosphonicacid can be prepared similar to the method described in French patent FR2669348 and by Y. Leroux et al., Phosphorus, Sulfur and Silicon, 63, 181(1991) for the preparation of hydroxydiphosphonic acid derivatives.According to such method, in a first step, an acid chloride of formula(II) ACOX is caused to react with a mixture of dimethylphosphite andtrimethylphosphite, and then in a second step, the ester functionsobtained in the previous step are hydrolyzed by acid hydrolysis,followed if necessary by a salification.

[0105] The acid chloride used in the first step is phenylacetyl chloride(C6H5-CH2-COCl) and the reaction is preferably carried out in a solventsuch as chloroform at a temperature below 30° C., for example at roomtemperature or at a temperature near 0° C., under neutral atmosphere,for example in a nitrogen atmosphere.

[0106] The hydrolysis of ester functions, in the second step, can becarried out by dissolving the product obtained in the first step inconcentrated hydrochloric acid, in excess, and refluxing during about 5to 15 hours.

[0107] Phenylacetic bisphosphonic acid prepared as indicated above isobtained with a very high yeld, higher than 95%.

[0108] Another object of the present invention relates to anypharmaceutical composition comprising in a pharmaceutically acceptablesupport at least one compound represented by formula (I) or (I′) such asdescribed hereinabove.

[0109] In an advantageous manner, this is a pharmaceutical compositionintended for the treatment or prophylaxis of disorders characterized byabnormal calcium metabolism, such as cancers or osteoporosis, whereinR₁, R₂ and R₃, which are the same or different, each represent ahydrogen atom, an alkyl, aryl, acyloxyalkyl or heterocycle group, and R′and R″, which are the same or different, each represent a hydrogen atom,an alkali metal or alkaline earth atom,

[0110] A represents a group with the formula —(CH₂)n-R₄,

[0111] R₄ represents a hydrogen atom, an alkyl group, an aryl group, aheterocycle or a group with the formula —NR₅R₆,

[0112] R₅ and R₆, which are the same or different, represent a hydrogenatom or an alkyl group,

[0113] n is a whole number from 0 to 24 inclusive,

[0114] their optical and geometrical isomers, their racemates, theirsalts, their hydrates and their mixtures.

[0115] The cancers are more specifically breast and prostate cancers andleukemias.

[0116] Malignant solid tumors are classified as carcinomas (95%) andsarcomas (5%). Development of carcinomas and sarcomas is correlated withvascularization of the tumor, known as angiogenesis. Any inhibition ofthis angiogenesis in the tumor makes it possible to shrink, or at leaststop or delay tumor growth. Now, it has been found in a surprisingmanner that compounds of the invention not only have an effect ontumors, but also an effect on angiogenesis.

[0117] The pharmaceutical composition according to the invention may beuseful notably for the treatment of malignant tumors, more specificallyfor the treatment of malignant solid tumors.

[0118] They may also be used to partially or totally inhibit tumorangiogenesis.

[0119] The pharmaceutical composition according to the invention mayalso be useful for the treatment of viral or inflammatory hepaticdiseases. Regarding this particular aspect, the pharmaceuticalcomposition comprises advantageously compounds of formula (I′) orphenylacetic bisphosphonicacid or salts thereof.

[0120] The invention is also directed to the method of treatment orprophylaxis in the human or animal body by administering to a subject,in particular a mammal and more specifically a human being, in need ofsuch treatment at least one compound of formula (I) or (I′) such asdefined hereinabove.

[0121] It further relates to a method of treating a disordercharacterized by abnormal calcium metabolism, such as cancers orosteoporosis, comprising administering to a subject, in particular apatient, in need of such treatment a therapeutically effective amount ofat least one compound represented by formula (I) or (I′) as definedabove, wherein R₁, R₂ and R₃, which are the same or different, eachrepresent a hydrogen atom, an alkyl, aryl, acyloxyalkyl or heterocyclegroup, and R′ and R″, which are the same or different, each represent ahydrogen atom, an alkali metal or alkaline earth atom,

[0122] A represents a group with the formula —CH₂)n-R₄,

[0123] R₄ represents a hydrogen atom, an alkyl group, an aryl group, aheterocycle or a group with the formula —NR₅R₆,

[0124] R₅ and R₆, which are the same or different, represent a hydrogenatom or an alkyl group,

[0125] n is a whole number from 0 to 24 inclusive,

[0126] their optical and geometrical isomers, their racemates, theirsalts, their hydrates and their mixtures.

[0127] Another object of the present invention is a method for treatingviral or inflammatory hepatic disease, comprising administering to apatient in need of such treatment a therapeutically effective amount ofat least one compound represented by formula (I) or (I′) as definedabove, wherein R₁, R₂ and R₃, which are the same or different, eachrepresent a hydrogen atom, an alkyl, aryl, acyloxyalkyl or heterocyclegroup, and R′ and R″, which are the same or different, each represent ahydrogen atom, an alkali metal or alkaline earth atom,

[0128] A represents a group with the formula —(CH₂)n-R₄,

[0129] R₄ represents a hydrogen atom, an alkyl group, an aryl group, aheterocycle or a group with the formula —NR₅R₆,

[0130] R₅ and R₆, which are the same or different, represent a hydrogenatom or an alkyl group,

[0131] n is a whole number from 0 to 24 inclusive,

[0132] their optical and geometrical isomers, their racemates, theirsalts, their hydrates and their mixtures.

[0133] With respect to these methods of therapeutic treatment andcompositions useful for these methods, and in a particular aspect, thecompounds are of formula (I) or (I′), with the exception of compounds offormula (I) wherein R₁, R₂ and R₃ simultaneously represent a hydrogenatom, and compounds represented by formula (I′) in which R′ and R″simultaneously represent a hydrogen atom.

[0134] Compounds according to the invention exhibit a specific activity,with no side effect and no toxicity, even in the case of a long lastingtreatment. Pharmacological and clinical studies have demonstrated thatcompounds of the present invention, for instance phenyl aceticbisphosphonic acid, has anti-tumor, anti-angiogenic and pro-apoptoticaffects, and that they are active both in vivo and in vitro against theproliferation of numerous tumor cell lines. For example, in vitro,phenylacetic bisphosphonic acid inhibits the proliferation of two breastcancer cell lines, representatives of two types of breast cancers (nonmetatasic hormonosensitive tumor and metastasic hormonoindependenttumor) even at a low dose, by breaking the cellular cycle and byinducing an apoptosis of tumor cells. In vivo, phenylaceticbisphosphonic acid irreversibly blocks the proliferation of MCF7-rascells and the growth of mammary tumors in the first five or six weeks oftreatment, and this effect is maintained without any toxic affect, evenwhen the treatment is applied during a long period of time. The in vivoand in vitro results thus demonstrate the proapoptotic andantiangiogenic effect of the compounds according to the invention. Suchassays are detailed in the examples thereafter.

[0135] Further, studies have demonstrated the action of phenylaceticbisphosphonic acid on hepatic inflammatory lesions induced by viruses.

[0136] These results show that phenylacetic bisphophonic acid and thepharmaceutically acceptable salts, partially esterified, or derivativesthereof can be efficiently used in the treatment of cancers and moreparticularly human breast or prostate cancer, and also in the treatmentof inflammatory hepatic diseases, such as acute and chronic hepatitis,for example viral or toxic hepatitis.

[0137] The antiangiogenic activity is also of interest in the treatmentof pathologic angiogenesis, for example diabetes retinopathies,rheumatoid polyarthritis and macular degeneration related to age.

[0138] The pharmaceutical compositions according to the inventionadvantageously comprise one or more pharmaceutically acceptableexcipients or vehicles. Some examples include saline, physiological,isotonic, buffered solutions, etc., compatible with pharmaceutical useand known to those skilled in the art. The compositions may contain oneor more agents or vehicles chosen among dispersives, solubilizers,stabilizers, preservatives, etc. The agents or vehicles that may be usedin the formulations (liquids and/or injectables and/or solids) comprisenotably methylcellulose, hydroxymethylcellulose, carboxymethylcellulose,polysorbate 80, mannitol, gelatin, lactose, vegetable oils, acacia, etc.The compositions may be formulated as suspensions for injection, gels,oils, tablets, suppositories, powders, capsules, gelules, etc., possiblyby means of pharmaceutical forms or devices allowing sustained and/ordelayed release. For this type of formulation, an agent such ascellulose, carbonates or starches is advantageously used.

[0139] The compounds or compositions according to the invention may beadministered in various ways and in different forms. For instance, theymay be injected by the systemic route or given orally, preferably by thesystemic route, such as, for example, by the intravenous, intramuscular,subcutaneous, transdermal, intra-arterial route, etc. For injections,the compounds are generally packaged as liquid suspensions, which may beinjected by means of syringes or infusions, for example. It isunderstood that the rate and/or dose injected may be adapted by thoseskilled in the art according to the patient, the pathology, the methodof administration, etc. Typically, the compounds are administered atdoses ranging from 0.1 μg to 500 mg/kg of body weight, more generallyfrom 0.01 to 10 mg/kg, typically between 0.1 and 10 mg/kg. Furthermore,repeated injections may be given as the case may be. Moreover, thecompositions according to the invention may additionally comprise otheractive substances or agents.

[0140] Other aspects and advantages of the present invention will becomeapparent from the following examples, which are given for purposes ofillustration and not by way of limitation.

[0141] General Bibliography on the Subject

[0142] Adami S. and Zamberlan N. (1996). Adverse effects ofbisphosphonates. A comparative review. Drug Saf., 14, 158-170.

[0143] Boissier S., Ferrerras M., Peyruchaud O., Magnetto S., EbetinoF., Colombel, Delmas P., Delaissé J M and Clézardin P. (2000).Bisphosphonates inhibit breast and cancer prostate carcinoma cellinvasion, an early event in the formation of bone metastases. CancerRes., 60, 2949-2954.

[0144] Boissier S., Magnetto S., Frappart L., Cuzin B., Ebetino F.,Delmas P. and Clézardin P. (1997). Bisphosphonates inhibit prostate andbreast carcinoma cell adhesion to unmineralized and mineralized boneextracellular matrices. Cancer Res., 57, 3890-3894.

[0145] Diel I., Solomayer E. and Bastert G. (2000). Bisphosphonates andthe prevention of metastasis: first evidences from preclinical andclinical studies. Cancer, 88, 3080-3088.

[0146] Ezra A., Hoffman A., Breuer E., Alferiev I. S., Monkkonen J., ElHanany-Rozen N., Weiss G., Stepensky D., Gati I., Cohen H., TormalehtoS., Amidon G. L., Golomb G. (2000). A peptide prodrug approach forimproving bisphosphonate oralabsorption. J Med Chem., 43, 3641-3652.

[0147] Fromigue O., Lagneaux L. and Body J. (2000). Bisphosphonatesinduce breast cancer cell death in vitro. J. Bone Miner. Res., 15,2211-2221. Paracrine and autocrine effect of vascular endothelial growthfactor: Inhibition of A431 tumor growth and angiogenesis byCarboxymethyl Benzylamide Dextran. Submitted to Cell Growth and Diff.

[0148] Hiraga T., Williams P., Mundy G. and Yoneda T. (2001). Thebisphosphonate ibandronate promotes apoptosis in MDA MB 231 human breastcancer cells in bone metastases. Cancer Res., 61, 4418-4424.

[0149] Ichinose Y., Migita K., Nakashima A., Kawakami A., Aoyagi T. andEguchi K. (2000). Effects of bisphosphonate on the release of MMP-2 fromcultured human osteoblasts. Tohoku J. Exp. Med., 192, 111-118.

[0150] Jagdev S., Coleman R., Shipman C., Rostami H. and Croucher P.(2001). The bisphophonate, zoledronic acid, induces apoptosis of breastcancer cells: evidence for synergy with paclitaxel. Br. J. Cancer, 84,1126-1134.

[0151] Lee M., Fong E., Singer R. and Guenette R. (2001). Bisphosphonatetreatment inhibits the growth of prostate cancer cells. Cancer Res., 61,2602-2608.

[0152] Lin J. H. (1996). Bisphosphonates: a review of theirpharmacokinetic properties. Bone,. 18, 75-85.

[0153] Lipton A. (2000) Bisphosphonates and breast carcinoma: presentand future. Cancer, 88, 3033-3037.

[0154] Luckman S. P., Hugues D., Coxon F., Graham R., Russell G. andRogers M. (1998). Nitrogen-containing phosphonates inhibit themevalonate pathway and prevent post-translational prenylation ofGTP-binding proteins, including Ras. J. Bone Miner. Res., 13, 581-589.

[0155] Mincey B., Moraghan T. and Perez E. (2000). Prevention andtreatment of osteoporosis in women with breast cancer. Mayo Clin. Proc.,75, 821-829.

[0156] Mondelo N., Peluffo V., Parma., Cointry G., Capozza R., FerrettiJ., Piccini E. and Montuori E. (1997). Preclinical toxicology ofbisphosphonates. Medicina (B. Aires), 57, 93-100.

[0157] Ogihara T., Kikuchi Y., Imai Y., Ohsaka A., Isaka M. and Oka Y.(1995). Acute lymphoblastic leukemia accompagnied by severehypercalcemia; successful treatment with bisphosphonate. RinshoKetsueki, 36, 29-34.

[0158] Pade V. and Stavchansky S. (1997). Estimation of the relativecontribution of the transcellular and paracellular pathway to thetransport of passively absorbed drugs in the Caco-2 cell culture model.Pharm. Res., 9, 1210-1215.

[0159] Patashnik S., Rabinovich L. and Golomb G. (1997). Preparation andevaluation of chitosan microspheres containing bisphosphonates. J. DrugTarget., 4, 371-380.

[0160] Perez-Atayde A., Sallan S., Tedrow U., Connors S., Allred E. andFolkman J. (1997). Spectrum of tumor angiogenesis in the bone marrow ofchildren with acute lymphoblastic leukemia. Am. J. Pathol., 150,815-821.

[0161] Ruifrok P. and Mol W. (1983). Paracellular transport of inorganicand organic ions across the rat ileum. Biochem. Pharmacol., 32, 637-640.

[0162] Senaratne S. G., Pirianov G., Mansi J. L., Arnett T. R. andColston K. W. (2000). Bisphosphonates induce apoptosis in human breastcancer cell lines. Br. J. Cancer, 82, 1459-1468.

[0163] Shipman C., Croucher P., Russell R., Helfrich M. and Rogers M.(1998). The bisphosphonate incadronate (YM 175) causes apoptosis ofhuman myeloma cells in vitro by inhibiting the mevalonate pathway.Cancer Res., 58, 5294-5297.

[0164] Shipman C., Rogers M., Apperley J., Russell R. and Croucher P.(1997). Bisphosphonates induce apoptosis in human myeloma cell lines: anovel anti-tumor activity. Br. J. Haematol., 98, 665-672.

[0165] Shipman C., Vanderkerken K., Rogers M., Lippitt J., Asosingh K.,Hughes D., Van Camp B., Russel G. and Croucher P. (2000a). The potentbisphosphonate ibandronate does not induce myeloma cell apoptosis in amurine model of established multiple myeloma. Br. J. Haematol., 111,283-286.

[0166] Takagi M., Takahiashi K., Maruyama T., Kaneko K., Obinata K.,Tadokoro R., Kastumata Miura Y., Fujita H., Ishimoto K. and Yabuta K.(1998). Acute lymphoblastic leukemia accompagnied by severe,hypercalcemia: successful treatment including aminohydroxypropylidenebisphosphonate (pamidronate disodium). Pediatr. Hematol. Oncol., 15,283-286.

[0167] Teronen O., Konttinen Y., Lindqvist C., Salo T., Ingman T.,Lauhio A., Ding Y., Santavirta S., Valleala H. and Sorsa T. (1997).Inhibition of matrix metalloproteinase-1 by dichloromethylenebisphosphonate (clodronate). Calcif. Tissue Int., 61, 59-61.

[0168] Van der Pluijm G., Vloedgraven H., Van Beek E., Van der Wee-PalsL., Lowik C. and Papapoulos S. (1996). Bisphosphonates inhibit theadhesion of breast cancer cells to bone matrices in vitro. J. Clin.Invest., 98, 698-705.

[0169] Van der Pluijm G., Sijmons B., Vloedgraven H., Deckers M.,Papapoulos S. and Lowik C. (2001). Monitoring metastatic behavior ofhuman tumor cells in mice with species-specific polymerase chainreaction: elevated expression of angiogenesis and bone resorptionstimulators by breast cancer in bone metastases. J. Bone Miner. Res.,16, 1077-1091.

[0170] Ylitalo R., Monkkonen J. and Yla-Herttuala S. (1998). Effectes ofliposome encapsulated bisphosphonates on acetylated LDL metabolism,lipid accumulation and viability of phagocyting cells. Life Sci., 62,413-422.

[0171] Yoneda T., Michigami T., Yi B., Williams P. J., Niewolna M. andHiraga T. (2000). Actions of bisphosphonate on bone metastasis in animalmodels of breast carcinoma. Cancer, 15, 2979-2988.

EXAMPLES

[0172] Unless otherwise indicated, percentages are expressed by weight.Y: yield.

[0173] Preparation of Compounds According to the Invention

[0174] Preparation of 1-hydroxy-1-phenylethylidene-1,1-bisphosphonicacid

[0175] To a mixture of trimethylphosphite (0.01 mole) anddimethylphosphite (0.01 mole) in 5 ml of chloroform, was added dropwisea solution phenylacetyl chloride in 5 ml of chloroform at 0° C., whilestirring the reaction mixture under dry nitrogen. The mixture is thenheated to 80° C. for about 8 hours.

[0176] After cooling, the mixture is washed and precipitated in ethylether. The white solid collected by filtration is the bisphosphonateester (1-hydroxy-1-phenylethylidene-1,1-bisphosphonate tetramethylester).

[0177] Yield: 97%

[0178] Melting point F=120° C.

[0179] NMR P³¹ (CDCl₃): δ=20.53 ppm

[0180] NMR H¹ (TMS, CDCl₃): δ=3.8 ppm; δ_(t(CH2))=3.4 ppm (³J_(HCCP):13.5 Hz); δ_(m(ph))=7.3 ppm.

[0181] The bisphosphonate ester obtained as indicated above isredissolved in a large excess of concentrated hydrochloric aqueous acidand then refluxed for 15 hours. The aqueous solution is washed withether, and then evaporated under vacuum on Rotavapor. The correspondingacid is thus obtained.

[0182] The acid is purified and reprecipitated in ether and in abenzene/ether mixture. The white precipitate is collected by filtrationto produce 1-hydroxy-1-phenylethylidene-1,1-bisphosphonic acid.

[0183] Yield: 98%

[0184] NMR P³¹ (D₂O): δ=19.53 ppm

[0185] NMR H¹ (D₂O) : δ_(t(CH2))=3.4 ppm (³J_(HCCP): 13.5 Hz);δ_(m(ph))=3.4 ppm.

[0186] Preparation of methyl-di(trimethylsilyl)phosphite

[0187] In a four-necked flask equipped with a mechanical stirrer,coolant, introduction ampoule, thermometer and nitrogen inlet valve areadded dropwise 100 ml of concentrated ammonia solution in 83 g ofdimethylphosphonate previously distilled if necessary. The solutionobtained is evaporated under reduced pressure. After repeatedco-evaporations with pyridine (100 ml) and benzene (2×100 ml), the whitesolid obtained is treated with 140 ml of hexamethyldisilazane. Theresulting mixture is refluxed for 6 hours. The solution is thendistilled to yield the expected compound.

[0188] Yield=58% BP 74-76 (20 mm Hg)

[0189]³¹P {1H} NMR (CDCl₃): δ=127.6 ¹H NMR (CDCl₃): δ=0.19(s,18H,Me₃Si), 3.30 (d,3H, J_(PH)=8 Hz, MeO).

[0190] Method of Synthesis of 1-hydroxymethylene-1,1-bisphosphonic acid.

[0191] One equivalent of acid chloride is placed in a three-necked flaskequipped with a magnet under an inert atmosphere (Ar). Two equivalentsof tris(trimethylsilyl)phosphite are added at room temperature withstirring. The solution is then left to stand for several minutes at roomtemperature. The hydrolysis is carried out in methanol at 25° C. for 1hour. Volatile fractions are evaporated under vacuum. The products arepurified by an ether wash.

[0192] (1-hydroxy-1-phosphono-ethyl)-phosphonic acid. White powder.Yield=98%.

[0193]³¹P {1H} NMR (D₂O): δ=19.4 ¹H NMR (D₂O): δ=1.48 (t, 3H, ³J=16 Hz,CH₂COH).

[0194] (1-hydroxy-1-phenyl-phosphono-ethyl)-phosphonic acid. Whitepowder. Yield=90%.

[0195]³¹P {1H} NMR (D₂O): δ=19.0. ¹H NMR (D₂O): δ=3.31 (t, 2H, ³J=14 Hz,CH₂COH), 7.26-7.32 (m, 4H, C₆H₅), 7.38 (t, 1H, ³J=7.5 Hz, C₆H₅).

[0196] (Hydroxy-phenyl-phosphono-methyl)-phosphonic acid. White powder.Yield=91%.

[0197]³¹P {1H} NMR (D₂O): δ=16.0. ¹H NMR (D₂O): δ=7.08-7.13 (m, 4H,C₆H₅), 7.46 (t, ³J=5 Hz, 1H, C₆H₅). ¹³C {1H} NMR (D₂O): δ=78.76 (t,¹J=145.75 Hz, COH), 128.9, 130.8, 131.2, 138.6 (C₆H₅).

[0198] (Hydroxy-p-nitrophenyl-phosphono-methyl)-phosphonic acid. Whitepowder. Yield=85%.

[0199]³¹P {1H} NMR (D₂O): δ=15.3. ¹H NMR (D₂O): δ=7.89 (d, 2H, ³J=8.5Hz, C₆H₄), 8.15(d, 2H, ³J=8.5 Hz, C₆H₄). ¹³C {1H} NMR (D₂O): δ=75.28 (t,¹J=149 Hz, COH), 124.67, 128.15;146.13, 148.35 (C₆H₄).

[0200] General Operating Procedure for Products of the TypeA—C(OH)PO(OH)₂PO(OMe)₂

[0201] In a four-necked flask equipped with a mechanical stirrer,coolant, introduction ampoule, thermometer and nitrogen inlet valve areadded dropwise 1 equivalent of trimethylphosphite in 1 equivalent ofacid chloride at 0° C. After a 2 hour reaction time at room temperature,the dimethyl α-ketophosphonate is completely formed (the reaction isfollowed by ³¹P NMR). One equivalent of tris(trimethylsilyl)phosphite isthen added dropwise. The solution is then stirred for 15 minutes.Methanol is then added and the solution stirred for 2 hours at roomtemperature. The solvent and volatile fractions are then evaporated. Theresulting product is a white powder which is purified by successivewashes with ether.

[0202] [(Dimethoxy-phosphoryl)-hydroxy-phenyl-methyl]-phosphonic acid

[0203] White powder. Yield=91%

[0204]³¹P {1H} NMR (D₂O): δ=23.58 (d, ²J=27 Hz) ; 12.92 (d, ²J=27 Hz).¹H NMR (D₂O): δ=7.73 (d, 2H, ³J=6.0 Hz, C₆H₅); 7.46-7.43(m, 3H, C₆H₅);3.79(d, 3H, ³J=10.0 Hz, OCH₃); 3.63 (d, 3H, ³J=10.0 Hz, OCH₃).

[0205] [(Dimethoxy-phosphoryl)-hydroxy-ethyl]-phosphonic acid

[0206] White powder. Yield=91%

[0207]³¹P {1H} NMR (D₂O): δ=29.1 (d, ²J=27 Hz); 19.90 (d, ²J=27 Hz). ¹HNMR (D₂O): δ=7.73 (d, 2H, ³J=6.0 Hz, C₆H₅); 7.46-7.43(m, 3H, C₆H₅);3.79(d, 3H, ³J=10.0 Hz, OCH₃); 3.63 (d, 3H, ³J=10.0 Hz, OCH₃).

[0208] General Operating Procedure for Products of the TypeA—C(OH)PO(OH)OMePO(OH)(OMe)

[0209] In a four-necked flask equipped with a mechanical stirrer,coolant, introduction ampoule, thermometer and nitrogen inlet valve areadded dropwise 2 equivalents of methyl-bis(trimethylsilyl)phosphite in 1equivalent of chloride. After a 10 minute reaction time at roomtemperature, the solution is methanolysed for 2 hours. The solvent andvolatile fractions are then evaporated. The resulting product is a whitepowder which is then purified by successive washes with ether.

[0210] Monomethyl ester of[hydroxy-(hydroxy-methoxy-phosphoryl)-phenyl-methyl] phosphonic acid.

[0211] White powder. Yield=90%

[0212]³¹P {1H} NMR (D₂O): δ=18,50 . ¹H NMR (D₂O): δ=7.43-7.41 (m, 2H,C₆H₅); 7.15-7.05 (m, 3H, C₆H₅); 3.79(d, 6H, ³J=6.0 Hz, OCH₃)

[0213] Monomethyl ester of [(gydroxy-(hydroxy-methoxy-phosphoryl)-ethyl]phosphonic acid

[0214] White powder. Yield=90%

[0215]³¹P {1H} NMR (D₂O): δ=23.9. ¹H NMR (D₂O): 2.96(d, 6H, ³J=6.0 Hz,OCH₃); 1.07-0.98 (m, 3H, CH₃).

[0216] Monomethyl ester of[gydroxy-(hydroxy-methoxy-phosphoryl)-2-phenyl-ethyl] phosphonic acid.

[0217] White powder. Yield=90%

[0218]³¹P {1H} NMR (D₂O): δ=20.79. ¹H NMR (D₂O): δ=7.26-7.11 (m, 5H,C₆H₅); 3.79(d, 6H, ³J=6.0 Hz, OCH₃); 3.15 (t, 2H,³J=10.0 Hz, C₆H₅—CH₂)

[0219] General Operating Procedure for Products of the TypeA—C(OH)PO(OH)(OMe)PO(OMe)₂

[0220] In a four-necked flask equipped with a mechanical stirrer,coolant, introduction ampoule, thermometer and nitrogen inlet valve areadded dropwise 1 equivalent of trimethylphosphite in 1 equivalent ofacid chloride at 0° C. After a 2 hour reaction time at room temperature,the dimethyl α-ketophosphonate is completely formed (the reaction isfollowed by ³¹P NMR). One equivalent of methylbis(trimethylsilyl)phosphite is then added dropwise. The solution isthen stirred for 15 minutes. Methanol is then added and the solution isstirred for 2 hours at room temperature. The solvent and volatilefractions are then evaporated. The resulting product is a colorless oil.It is purified by successive washes with ether.

[0221] Dimethyl ester of[hydroxy-(hydroxy-methoxy-phosphoryl)-phenyl-methyl]-phosphonic acid

[0222] White powder. Yield=90%.

[0223]³¹P {1H} NMR (CDCl₃): δ=16.74 (d, ²J=47 Hz); 14.52 (d, ²J=47 Hz).¹H NMR (D₂O): δ=8.60-8.40 (s, 2H, OH); 7.49 (d, 2H, ³J=8.0 Hz, C₆H₅);7.04-6.97(m, 3H, C₆H₅); 3.47(d, 3H, ³J=10.0 Hz, OCH₃); 3.34 (d, 3H,³J=10.0 Hz, OCH₃); 3.23 (d, 3H, ³J=10.0 Hz, OCH₃).

[0224] Dimethyl ester of[hydroxy-(hydroxy-methoxy-phosphoryl)-ethyl]-phosphonic acid

[0225]³¹P {1H} NMR (CDCl₃): δ=20.46 (d, ²J=48 Hz) ; 18.67 (d, ²J=48 Hz).¹H NMR (D₂O): δ=8.98 (s, 2H, OH) 3.92(d, 3H, ³J=10.0 Hz, OCH₃); 3.88 (d,3H, ³J=10.0 Hz, OCH₃); 3.86 (d, 3H, ³J=10.0 Hz, OCH₃); 1.67 (t,3H,³J_(H-P)=15.5 Hz).

[0226] Anti-Proliferative and Anti-Invasive Activities of the CompoundsAccording to the Invention

[0227] Two bisphosphonates designated as BP1 and BP2 whose formulas areshown below were tested in cell and animal models. BP1 was not partiallyesterified and serves as a comparative example.

[0228] In vitro Tests

[0229] Materials and Methods:

[0230] The bisphosphonates BP1 and BP2 were tested in different celllines:

[0231] FRCjun MRA, a murine fibrosarcoma cell line produced bytransfection of FR3T3 cells by the oncogene jun-4,

[0232] A 431 human carcinoma cells. These cells harbor a VEGF (vascularendothelial growth factor) autocrine loop and, after implantation inimmunosuppressed mice, induce rapid development of highly angiogenictumors.

[0233] MDA MB 435 human breast tumor cells,

[0234] HUV-EC-C, transformed human umbilical cord endothelial cells(HUVEC),

[0235] BBC, bovine cerebral capillary endothelial cells.

[0236] Cell proliferation in the presence of different concentrations ofBP1 or BP2 was measured by an MTT test after 24, 48 and 72 h of culture.

[0237] In vitro Test Results:

[0238] The bisphosphonates (0.1-2 mM) inhibit the proliferation ofFRCjunMRA, MDA MB 435 and A 431 cells in a time- and dose-dependentmanner (FIGS. 2 and 3). For example, BP2 (2 mM) inhibits theproliferation of the three cell lines by 40%, 60% and 30%, respectively,after 48 h of culture. After 72 h in the presence of BP2 (2 mM),proliferation of A 431 cells is inhibited by 80% (P<0.05) (FIG. 3).

[0239] A trypan blue exclusion test shows that BP1 and BP2 do not inducecytotoxicity, even at concentrations above 5 mM.

[0240] Furthermore, the bisphosphonates (0.1-2 mM) also inhibit theproliferation of HUV ECC and BBC endothelial cells in a time- anddose-dependent manner.

[0241] Toxicity Tests

[0242] Tumor-free immunosuppressed mice were treated with BP1 and BP2 atdoses ranging from 0.06 to 6 mg per injection and per animal. Micereceived one injection per day for one week. Animals were weighedbefore, during and after treatment.

[0243] BP1 doses less than 6 mg/injection and all BP2 doses tested(0.06-6 mg/injection) caused no signs of toxicity such as hair loss,diarrhea, infection or anemia in the treated animals.

[0244] In vivo Tests

[0245] Materials and Methods:

[0246] A 431 cells were injected subcutaneously into immunosuppressedmice. The mice developed tumors within one week. Tumor-bearing mice wereassigned randomly to a control group or to treatment groups receivingdifferent concentrations of BP1 and BP2.

[0247] Mice received twice weekly by subcutaneous injection near thetumor either 0.1 ml of PBS alone for the controls (n=6), or PBScontaining either BP1 or BP2 at the following doses: 0.006 (n=6); 0.06(n=6) or 0.6 mg/injection (n=6). Tumors were measured weekly and theirvolume V calculated by the formula:

V=(4/3)πR1² R2 wherein R1 is radius 1 and R2 radius 2, with R1<R2.

[0248] Animals were sacrificed and the tumors excised, then weighed,fixed, embedded in paraffin and sliced. Endothelial cells from tumorblood vessels laid down during tumor angiogenesis were visualized byimmunohistochemical methods (GSL 1). Tumor angiogenesis was evaluated byimage analysis with NIH image software.

[0249] In vivo Test Results

[0250] Treatment with bisphosphonates inhibited the growth of A 431tumors and was maximal even at the lowest doses (FIG. 4). After 5 weeksof treatment, BP1 and BP2 (0.6 mg/injection/mouse) inhibited the growthof A 431 tumors by 40% and 56%, respectively (P<0.05).

[0251] In contrast to the in vitro results, the in vivo results showthat BP2 has greater efficacy than BP1.

[0252] Furthermore, inhibition of the growth of A 431 tumors in thetreated animals was correlated with inhibition of intratumoralangiogenesis (FIG. 5). Such inhibition, demonstrated by a significantdecrease in endothelial cell area per unit of area, was observedstarting from the lowest doses of BP1 and BP2.

[0253] To our knowledge, the synthesized bisphosphonates are the firstto show antitumoral action in vivo on the primary tumor, their effectsbeing correlated with an inhibition of angiogenesis.

[0254] In vitro Study of Sodium Phenylacetate Bisphosphonate

[0255] The study of the effect of sodium phenylacetate bisphosphonate oncell growth and cell viability has been carried out as indicatedhereafter.

[0256] MCF7-ras cells are cultivated (20,000 cells/well), on a dish of24 wells. After incubation for 24 hours, the medium (DMEM with 10%foetal calf serum) is replaced with the same medium to which are addedvarious concentrations of phenylacetate bisphosphonate (2 mM, 4 mM, 6mM, 8 mM and 10 mM). The cells are incubated for 1 to 4 days at 37° C.The cell viability is controlled by the Trypan blue test.

[0257] It is then noticed that phenylacetate bisphosphonate iscytostatic for the three first days of culture when concentrations arelower than 6 mM. For higher concentrations (8 et 10 mM) a slighttoxicity (10%) is observed, which becomes more important (50-60%) by thefourth day of culture.

[0258] In order to check the reversibility of the effect ofphenylacetate bisphosphonate, MCF7-ras cells have been treated withincreasing concentrations of phenylacetate bisphosphonate for 10 days,the medium being changed every two days. The cells are divided in twogroups. The DMEM medium supplemented with 10% of foetal calf serum isadded to the first group, and the same medium with 10 mM ofphenylacetate bisphosphonate is added to the second group, the mediumbeing changed every two days. After 15 days, tritiated thymidine isadded during 4 hours and the radioactivity of the suspension isdetermined by using a beta liquid scintigraphic counter (Beckman).

[0259] An inhibition of the cells proliferation is observed which ispartially reversible at the dose of 2 mM, and which is irreversible at adose above 4 mM.

[0260] Proapoptotic Activity of Sodium Phenylacetate Bisphosphonate

[0261] In order to determine the nature of the toxicity of sodiumphenylacetate bisphosphonate, MCF7 and MCF7-ras cells are cultivated,and then washed after 24 hours, and cultivated for 3 hours in DMEMmedium with 10% of foetal calf serum. Phenylacetate bisphosphonate (10mM) is added to one group but not to the other group. Apoptosis isdetermined by the test of Annexine V conjugated with FITC antibody.Propidium iodide is used in order to determine early apoptosis (positivestaining for Annexine V and negative staining with propidium iodide) andlate apoptosis (positive staining with Annexine V and with propidiumiodide).

[0262] After 4 hours of treatment with phenylacetate bisphosphonate, thepercentage of cells in early apoptosis is almost identical in the twocell types, whilst the percentage of cells in late apoptosis is moreimportant with MCF7-ras cells (22% and 53% respectively).

[0263] The cell apoptosis is confirmed by the method of DNA degradation.MCF7 and MCF7-ras cells are cultivated (5×10⁵ cells in T25 flasks).After 24 hours, the cells are washed, and then cultivated in DMEM mediumwith 10% of foetal calf serum ; phenylacetate bisphosphonate (10 mM) isadded to one group, and no phenylacetate bisphosphonate is added to theother group.

[0264] After 96 hours, the cell extract containing the fragmented DNA isincubated with 0.5 mg/ml of RNase A at 37° C. for one hour, then with0.5 mg/ml of proteinase K for one hour at 37° C. After incubation, thefragmented DNA is precipitated by isopropanol and then dissolved in 10mM of Tris-HCl (pH 8) 1 ml pf EDTA, 5% of glycerol and 0.05% bromophenolblue. The fragmented DNA separated by electrophoresis on agarose gel at1% is marked with ethidium bromide and photographed in UV.

[0265] A dramatic degradation of DNA of MCF7 and MCF7-ras cells is thenobserved in the group containing 10 mM of phenylacetate bisphosphonate.

[0266] Theses results demonstrate that phenylacetic bisphosphonic acidinhibits the proliferation and induce the apoptosis MCF7 and MCF7-rascells.

[0267] In vivo Study of Sodium Phenylacetate Bisphosphonate

[0268] The antitumor effect of sodium phenylacetate bisphosphonate isverified in mice.

[0269] Inoculation of 4×10⁶ MCF7-ras cells in female athymic nude miceis made sub-cutaneously in a 0.1 ml volume of DMEM, which induces 70% oftumors after three weeks.

[0270] Treatment with phenylacetate bisphosphonate begun when the meanvolume of the tumors was 550 mm³. The animals are divided in two groups,on receiving phenylacetate bisphosphonate, and the other no.Phenylacetate bisphosphonate is injected sub-cutaneously andperitumorally, twice a week, during 5 weeks, and the doses are 80 mg/kg(5 cases) and 160 mg/kg (6 cases).

[0271] At the end of the treatment, an inhibition of the growth of thetumors is observed; it is irreversible and dose dependent. When thetreatment is stopped, the growth of the tumors is blocked for at leastthree weeks in the case of the mice treated with a 160 mg/kg dose,whilst tumor growth relapsed is observed in the case of mice treatedwith a 80 mg/kg dose.

[0272] The immunohistologic analysis of the tumors show that thedecrease in tumor growth is related to induction of apoptosis, to asharp decrease of angiogenesis of the tumor and induction of fibrose inthe tumor.

[0273] Pro-apoptotic Effet

[0274] Further, the proapoptotic effect of phenylacetate bisphosphonateis confirmed by the technique of antibody staining. The staining ofapoptotic cells is made by the M30 antibody which recognises thespecific site of caspases on the cytokeratine 18 filaments produced bythe epithelial cells. Such filaments rapidly aggregate in the cells inearly apoptosis, by hyperphosphorylation of cytokeratines. By thiscytoplasmic staining, granular structures are observed inside thecytoplasm, which corresponds to cells in late apoptosis.

[0275] These granular structures are observed in the tumor which havebeen treated with a high dose (160 mg/kg) of phenylacetatebisphosphonate. Further, an increase in the staining withanticytokeratine 18 antibody with concentration dose-dependently isnoticed. By this way, early apoptosis is clearly distinguished from lateapoptosis by the presence of granular structures in the cytoplasm.

[0276] This confirms that the treatment of tumors with phenylacetatebisphosphonate with a 80 mg/kg dose results in a apoptosis whilst thetreatment with a 160 mg/kg dose results in a aponecrosis, which is anintermediate state between apoptosis and necrosis.

[0277] Anti-angiogenic Effect

[0278] The anti-angiogenic was verified by the GSL1 lectin method.

[0279] By using GSL1 lectin, angiogenesis is determined and identifiedby the red coloration staining of endothelial cells with anti-GSL1antibody. It is noted that this angiogenesis is inhibited by thetreatment of the tumors with phenylacetate bisphosphonate. This effectis partial but important (80%) at the 80 mg/kg dose. and complete at 160mg/kg.

[0280] These assays demonstrate that phenylacetic bisphosphonic acidexhibits a very important antiangiogenic effect, and also a antitumorand proapoptotic effect. These results confirm that phenylaceticbisphosphonic acid is a potentially active medicament for the treatmentof cancer tumors, more particularly breast cancer.

[0281] Effect on Inflammatory Hepatic Cells

[0282] The study is carried out on nude mice having an acute hepatitis.The mice are divided in two groups, one being treated with sodiumphenylacetate bisphosphonate, whilst the second is not. Phenylacetatebisphosphonate is administered to the treated mice sub-cutaneously (80mg/kg or 160 mg/kg), twice a week for five weeks. The mice aresacrificed and the liver is histologically analyzed after HES (method ofHematoxiline Eosine Staining).

[0283] Examination with optical microscope shows that the livers of thenon-treated mice is seriously damaged by comparison with the treatedmice. More particularly, necrotic areas resulting from chronic hepatitisare noted, and also liquids accumulation in the hepatocytes (steatose)associated with a lymphocyte cells inflammation. Such hepatic conditionresults in the hepatic tissues necrosis, and the formation of thefibrous area. In contrast, in the case of the mice treated withphenylacetate bisphosphonate, in 4 cases out of 5, hepatic toxicity hasbeen noted.

1-9 (Cancelled)
 10. (Currently Amended) Method of preparation of compounds represented by formula (I):

[[,]] wherein the method it comprises the following steps: contacting at least one acid halide represented by formula (II): ACOX, or one α-ketophosphonate represented by formula (III):

with at least one silyl phosphite represented by formula (IV): P[(OSialk₃)_(x)][OR₃]_(3-x)  (IV) wherein R₁, R₂ and R₃, which are the same or different, represent an alkyl, aryl, acyloxyalkyl, or heterocycle group, A being defined in one of the previous claims represents a group of formula —(CH₂)_(n)—R₄, X represents a halogen atom, preferably chlorine, alk is a C1-6 alkyl group, x is equal to 2 or 3, n is a whole number from 0 to 24 inclusive, R₄ represents a hydrogen atom, an alkyl group, an aryl goup, a heterocycle or a group with the formula —NR₅R₆, R₅ and R₆, which are the same or different, represent a hydrogen atom or an alkyl group, hydrolysis of the compounds obtained in the previous step.
 11. (Original) Method according to claim 10, wherein it comprises the following steps: contacting a least one acid halide represented by formula (II): ACOX, with at least one phosphite represented by formula (V): P(OR1)(OR2)(OR), wherein R₁, R₂ and R, which are the same or different, represent an alkyl, aryl, acyloxyalkyl, or heterocycle group, to form an α-ketophosphonate represented by formula (III), contacting the α-ketophosphonate obtained in the previous step with at least one sylyl phosphite represented by formula (IV) such as defined in the preceding claim, hydrolysis of the compounds obtained in the previous step.
 12. (Original) Method according to claim 11, wherein the silyl phosphite is a compound represented by formula (IV) in which x is equal to
 2. 13. (Original) Method according to claim 11, wherein the silyl phosphite is a compound represented by formual (IV) in which x is equal to
 3. 14. (Original) Method according to claim 10, wherein at least one acid halide represented by formula (II): ACOX, is placed in contact with at least one silyl phosphite represented by formual (IV) in which x is equal to 2, then the product so obtained is placed in contact with at least one silyl phosphite represented by formual (IV) in which x is equal to
 3. 15. (Currently Amended) Method of preparation of compounds represented by formula (I′):

[[,]] wherein the method it comprises the following steps: an acid chloride of formula (II): ACOX is caused to react with a mixture of dimethylphosphite and trimethylphosphite, wherein A represents a group of formula —(CH₂)_(n)—R₄, X represents a halogen atom, preferably chlorine, n is a whole number from 0 to 24, R₄ represents a hydrogen atom, an alkyl group, an aryl group, a heterocycle or a group with the formula —NR₅R₆, R₅ and R₆, which are the same or different, represent a hydrogen atom or an alkyl group, and R′ and R″, which are the same or different, each represent a hydrogen atom, an alkali metal or alkaline earth atom; and then in a second step, the ester functions obtained in the previous steps are hydrolyzed by acid hydrolysis, followed by a salification.
 16. (Original) Method according to claim 15, wherein the reaction of the first step is carried out in a solvent such as chloroform at a temperature below 30 ° C.
 17. (Original) Method according to claim 15, wherein the hydrolysis is carried out by dissolving the product obtained in the first step in concentrated hot hydrochloric acid. 18-25 (Cancelled) 